Tissue plasminogen activator (tPA) thrombolysis is beneficial for thrombotic stroke, but its direct neuronal and vascular toxicities are problematic. Our extensive pilot data suggest that: (1) protein S (PS) has antithrombotic and neuroprotective activities in mouse stroke models;(2) PS protects neurons and brain endothelial cells (BEC) from N-methyl-D-aspartate (NMDA) and oxygen/glucose deprivation (OGD) injuries;(3) PS's cytoprotection requires its C-terminal sex hormone binding globulin-like (rSHBG) module, but not the N-terminal micro-PS [Gla domain, thrombin-sensitive (TSR) region, EGF1 module];(4) PS activates the PISK-Akt cell survival axis and inhibits the intrinsic apoptotic cascade in NMDA-treated neurons and OGD- treated BEC and the extrinsic apoptotic cascade in tPA/NMDA-treated neurons and tPA/OGD-treated BEC;and (5) PS's cytoprotection may require the rse/Tyro-3 receptor tyrosine kinase (RTK), but not the Axl/Tyro 7 or Mer/Tyro 12 RTKs. Thus, we hypothesize that PS and certain of its variants may protect brain from ischemic/thrombotic events by antithrombotic activity involving Gla, TSR and EGF1 modules, and by direct cytoprotection involving the SHBG domain. We further hypothesize that combined therapies for stroke with tPA and PS, rSHBG and the GlaFII PS mutant will directly protect brain cells because PS and its variants with intact SHBG domain can activate the Tyro 3-Akt cell survival signaling pathway, and because this cytoprotective action can compensate for tPA's cytotoxicities. The research design proposes to test the hypothesis: first, using mechanical and embolic mouse stroke models (aim 1);second, using an in vivo mouse model of NMDA and tPA excitotoxic brain lesions to isolate intravascular PS and tPA effects from their direct effects on brain cells (aim 2);and third, using in vitro models of NMDA and tPA/NMDA neuronal injuries (aim 3) and OGD and tPA/OGD BEC injuries (aim 4). Expertise and reagents provided by collaborators/consultants will be Dr. J.H. Griffin (Scripps) (PS reagents, mouse tPA), Dr. G. Lemke (Salk) (Tyro 3 RTK mutants), Dr. R. Freeman (Rochester) (various Akt, Bel and FKHRL1 mutants), Dr. M. Moskowitz (Harvard) (intrinsic/extrinsic apoptotic signaling), Dr. B. Berk (Rochester) (Tyro 3 RTK-Akt signaling) and Dr. M. Chopp (Henry Ford) (rat stroke model). The results will provide mechanistic insights and test therapeutic agents that may be translated to improved therapy for ischemic stroke.